EMBO/SourceData huggingface dataset:
Figure 1Survival curves for control (n=23 biological replicates), Rorc(t)CreTg Tet1/3fl/fl(n=20 biological replicates), Rorc(t)CreTg Tet1/2/3fl/fl mice (n=10 biological replicates). Paired male or female mice were used and no gender biases associated with genotypes were observed.Representative H&E staining of the lungs (top panel) and small intestine (bottom panel) and histopathological scoring of 3 months old control (n=3 biological replicates) and Rorc(t)CreTg Tet1/3fl/fl mice (n=3 biological replicates). Data are means +/-SEM, One-Way ANOVA with uncorrected Fisher's LSD test. Scale bar = 263 and 53 µm, top and bottom panels, respectively. , Paired male or female mice were used and no gender biases associated with genotypes were observed.Frequency of FoxP3+ cells among CD4+ T cells in the thymus (n=6 or 10 biological replicates), peripheral lymph nodes (n=5 or 6 biological replicates), spleen (n=5 or 6 biological replicates) and mesenteric lymph nodes (n=4 or 8 biological replicates) pre-gated on live CD4+TCRβ+ T cells and representative flow plots (bottom). Data are means +/- SEM, One-Way ANOVA with uncorrected Fisher's LSD test. Paired male or female mice were used and no gender biases associated with genotypes were observed.Absolute number of FoxP3+ Tregs from the thymus (n=5 or 7 biological replicates) and spleen (n=7 or 8 biological replicates) of control Tet1/3fl/fl and Rorc(t)CreTg Tet1/3fl/fl mice. Data is means +/- SEM, unpaired Mann-Whitney t test. Paired male or female mice were used and no gender biases associated with genotypes were observed.Quantification of CD25 MFI expression on FoxP3+ T cells in the spleen of control Tet1/3fl/fl (n=4 biological replicates) and Rorc(t)CreTg Tet1/3fl/fl mice (n=4 biological replicates). Data is means +/- SEM, unpaired Mann-Whitney t test. Paired male or female mice were used and no gender biases associated with genotypes were observed.Quantification of FoxP3 MFI expression on FoxP3+ T cells in the spleen of control Tet1/3fl/fl (n=6 biological replicates) and Rorc(t)CreTg Tet1/3fl/fl mice (n=6 biological replicates). Data is means +/- SEM, unpaired Mann-Whitney t test.   Quantification of ICOS MFI expression on FoxP3+ T cells from the mesenteric lymph nodes of control Tet1/3fl/fl (n=5 biological replicates) and Rorc(t)CreTg Tet1/3fl/fl (n=7 biological replicates) mice. Data is means +/- SEM, unpaired Mann-Whitney t test.   , Paired male or female mice were used and no gender biases associated with genotypes were observed.
Figure 2Representative flow plots, histogram and quantification of CD4 gMFI expression in Treg cells activated as in (A). Data is means+/-SEM, unpaired t-test.Weight curve of female Rag-/- mice after T-cell transfer colitis induced via transfer of naïve WT CD45.1 conventional CD4+ T cells alone (n=5 recipients) or in combination with Tregs isolated from Tet1/3fl/fl and Rorc(t)CreTg Tet1/3fl/fl littermates (n=5 recipients/group). Data is means +/- SEM, Two-Way ANOVA with Geisser-Greenhouse correction. Experiment was replicated 2 times.Representative flow plots and quantification of percent Tbet+, Rorγt+ and FoxP3+ of CD45.1 CD4+ effector cells isolated from the MLN of female Rag-/- mice 7 weeks post T cell transfer induced colitis. Respective cohorts were Naïve CD45.1 T cells only (n=5 mice), WT CD45.1+ Tet1/3fl/fl Tregs (n=5 mice) and WT CD45.1+Rorc(t)CreTg Tet1/3fl/fl Tregs (n=4 mice). Cells were pre-gated on Live CD45.1+TCRβ+ cells. Data is means+/- SEM, Two-Way ANOVA with Tukey's Multiple comparison test. Experiment was replicated 2 times.
Figure 3Quantification of Live CD4+ FoxP3+ T cells absolute cell numbers in different organs of FoxP3Cre Tet1/3+/+ (n=3 biological replicates) and FoxP3Cre Tet1/3 fl/fl (n=3 biological replicates) male littermate mice. Data is shown as means+/- SEM, One-Way ANOVA with uncorrected Fisher's LSD test.Quantification of CD25-FoxP3lo, CD25+FoxP3- and CD25+FoxP3+ cells among Live CD73-CD4+ T cells thymocytes of control Tet3fl/fl (n=5 biological replicates) and Rorc(t)CreTg Tet3fl/fl mice (n=3 biological replicates) mice. Data is shown as means+/- SEM, Two-Way ANOVA with Sidak's multiple comparison test. Quantification of CD25-FoxP3lo, CD25+FoxP3- and CD25+FoxP3+ cells among Live CD73-CD4+ T cells thymocytes of control Tet1fl/fl (n=6 biological replicates) and Rorc(t)CreTg Tet1fl/fl mice (n=6 biological replicates). Data is shown as means+/- SEM, Two-Way ANOVA with Sidak's multiple comparison test. paired male or female mice were used and no gender biases associated with genotypes were observed.Quantification of Nur77 staining among TCRβhi CD69+ CD4 SP thymocytes from control Tet3fl/fl (n=5 biological replicates) and Rorc(t)CreTg Tet3fl/fl mice (n=3 biological replicates). Data is as means+/- SEM, unpaired two-tailed Mann-Whitney t-test Quantification of Nur77 staining among TCRβhi CD69+ CD4 SP thymocytes from control Tet1fl/fl (n=4 biological replicates) and Rorc(t)CreTg Te1fl/fl mice (n=3 biological replicates). Data is shown as means+/- SEM, unpaired two-tailed Mann-Whitney t-test paired male or female mice were used and no gender biases associated with genotypes were observed.
Figure 4Quantification of % FoxP3+ cells 72hrs post activation of sorted naïve CD4+ T cells with anti-CD3/CD28 and 20 ng/mL TGF-β in the presence of absence of indicated amounts of rhIL-2 for 72hrs (n=3 biological replicates per dose). Data is shown as means+/-SEM of 3 independent samples, Two-Way ANOVA with uncorrected Fisher's LSD test.IL-2 mRNA quantification by qPCR in CD4+ T cells post-activation at indicated time-points (n=2 biological replicates/genotype). Cells were stimulated with anti-CD3/CD28 without exogenous IL-2 or TGF-β. Data is shown as means+/-SEM, Two-Way ANOVA with Sidak's multiple comparison testIL-2 quantification by ELISA in supernatants collected at 48 hours post activation of naïve CD4+ T cells with anti-CD3/CD28 without exogenous IL-2 and in the presence or absence of 20ng/mL TGF-β (n=4 biological replicates). Data is shown as means+/-SEM, Two-Way ANOVA with Bonferroni's multiple comparison testIL-2 quantification by ELISA in supernatants collected at 5 hours post activation of naïve CD4 T cells with PMA/Ionomycin (n=7). Data is shown as means+/-SEM, Two-Way ANOVA with Sidak's multiple comparison test   IL-2 quantification by ELISA in supernatants collected at 72hrs following activation of CD4 T cells with anti-CD3/CD28 without exogenous IL-2 or TGF-β (n=3). Data is shown as means+/-SEM, One-Way ANOVA with Tukey's Multiple comparison test.  Quantification of % FoxP3+ cells among CD4 T cells 72hrs activation with anti-CD3/CD28 and 20 ng/mL TGF-β in the presence or absence of 50U rhIL-2 for 72hrs (n=3 biological replicates). Data is shown as means+/-SEM, One-Way ANOVA with Tukey's Multiple comparison test.   Quantification of % FoxP3+ cells among CD4 T cells 72hrs activation with anti-CD3/CD28 and 20 ng/mL TGF-β in the presence or absence of 50U rhIL-2 for 72hrs (n=3 biological replicates). Data is shown as means+/-SEM, One-Way ANOVA with Tukey's Multiple comparison test.
Figure 5Integrated genome viewer (IGV) browser tracks of the Lck, Fyn gene loci highlighting differential accessible regions (in red boxes) in biological replicates of CD4+ T cells activated with anti-CD3/CD28 and 20 ng/mL TGF-β +/- 100U IL-2.Integrated genome viewer (IGV) browser tracks of the Itk gene loci highlighting differential accessible regions (in red boxes) in biological replicates of CD4+ T cells activated with anti-CD3/CD28 and 20 ng/mL TGF-β +/- 100U IL-2.(G) IGV browser track of the IL-2 locus showing accessibility peaks at the IL-2 promoter region and upstream of the promoter (red arrow) in biological replicates of CD4+ T cells activated as in (B).
Figure 1B Cryosections were subjected to immunofluorescence microscopy using appropriate excitation wavelengths and emission filters. White boxes indicate the magnified area in the lower panel. All images were taken as "multiple image alignments". Scale bars represent 100 and 25 µm in the upper and lower panel, respectively.C Whole brain and midbrain lysates of adult mice were prepared as outlined ;. Total protein (20 µg) was immunoblotted for MAD2 or α-Tubulin and compared to HEK-293 cell lysates.D Z-stacks of individual TPH+/MAD2+ positive neurons were generated by confocal microscopy. Intensity profiles of an average projection were generated using ImageJ/Plot Profile software. Scale bar represents 20 µm.
Figure 2B - D GST pull-down experiments were conducted in duplicates using the indicated GST fusion proteins. Eluates (30%) were subjected to immunoblotting together with an aliquot (1%) of the input. Blots were stained with Ponceau S and MAD2 was detected by immunoblotting. Densitometric signals were quantified using ImageJ software. Results were compared using unpaired two-tailed t-tests; ****p < 0.0001, ns - not significant; exact p-values are indicated; error bars represent SD of 8 independent biological replicates (n = 8).E Total cell lysates (5 µg) of indicated transfections were subjected to immunoblotting, using antibodies against YFP and MAD2.YFP-hMAD2-constructs were generated Whole cell lysates of transfected HEK-293 cells were used for GST pull-down experiments. One-way ANOVA with Dunnett's multiple posthoc comparison; ***p < 0.001, ns - not significant; error bars represent SD of 3 independent biological replicates (n = 3).
Figure 3A - B Immunofluorescence of p31comet and BubR1 in mouse dorsal raphe. White boxes indicate the magnified sections in the lower panels. Scale bars represent 100 and 25 µm in the upper and lower panels, respectively.C Co-immunoprecipitation experiments using lysates from HEK-293 cells stably expressing the indicated YFP-tagged transporters. Eluates (20%) as well as 0.1% total cell lysate were analyzed by immunoblotting. The immunoblot for mouse anti-β2-adaptin was subsequently used for incubation with rabbit anti-GFP primary and IRDye 800CW anti-rabbit secondary antibody. IRDye signals were detected using the LI-COR Odyssey CLx imaging system. Densitometric signals were quantified using ImageJ software.HEK-293 cells stably expressing YFP-hSERT were transfected with indicated siRNAs ("C" - ctrl siRNA; "M" - MAD2 siRNA) and subjected to co-immunoprecipitation. All signals were normalized to YFP-SERT. Results were compared using unpaired two-tailed t-tests; ***p < 0.001, ****p < 0.0001, ns - not significant; calculated p-values are indicated; error bars represent SD of 7 (BubR1), 8 (β2-adaptin), 5 (p31comet) and 8 (HSP70) independent biological replicates.
Figure 4A Radioactive substrate uptake was conducted KM and Vmax were determined by fitting the data to the equation of a rectangular hyperbola. Representative curves are shown. Error bars represent SEM. For comparison of Vmax paired two-tailed t-test was conducted; **p < 0.01, ns - not significant; exact p-value is indicated. Data are derived from 6 independent biological replicates. Lysates were prepared from an aliquot of the cells, which were used for the uptake assay, and subjected to immunoblotting to verify MAD2 knockdown. Immunodetection of α-tubulin was used as a loading control.B Cell surface biotinylation using HEK-293 cells stably expressing YFP-hSERT was performed Whole cell lysates and streptavidin-conjugated fractions (i.e., surface protein) were subjected to immunoblotting.C - D HEK-293 cells stably expressing YFP-hSERT were transfected with the indicated siRNAs, imaged and analyzed Representative maximum intensity projections of Z-stacks are shown. Scale bars represent 50 and 20 µm in full size images and zoomed areas, respectively. Intracellular SERT-signal was measured using ImageJ software. The dot plot in panel D shows values (presented as Log10 to account for the large variation in the signal) from individual cells (Ctrl siRNA n = 112 cells; MAD2 siRNA n = 116 cells) deriving from 3 independent biological replicates, the median and the interquartile range. The statistically significant difference was assessed by an unpaired two-tailed t-test, the p-value is indicated.E - H HEK-293 cells stably expressing YFP-hSERT were transfected with the indicated siRNA, subsequently co-transfected with plasmids encoding mCherry-Rab5 (panel E) or mCherry-Rab7A (panel G) Manders' overlap coefficients for the fraction of mCherry overlapping YFP were calculated for multiple individual images using ImageJ/JACoP. Number of analyzed images: mCh-Rab5/ctrl-siRNA = 38; mCh-Rab5/MAD2-siRNA = 33 (Panel F); mCh-Rab7A/ctrl-siRNA = 13; mCh-Rab7A/Mad2-siRNA = 14 (panel H). Statistically significant differences were assessed by unpaired two-tailed t-test, the p-values were below 0.0001. Error bars represent SD of 3 independent biological replicates (n = 3). Scale bars represent 50 and 20 µm in full size images and zoomed areas, respectively.
Figure 5B Immunofluorescence images of rat primary dorsal raphe neurons were analyzed using ImageJ/JACoP software. Bars indicate Manders' overlap coefficients for the fraction of SERT overlapping with the denoted marker protein. One-way ANOVA and Tukey's multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Number of analyzed cells: Rab11A n = 20; Rab7A n = 11; Rab4 n = 8; LAMP1 n = 12; Rab5 n = 14; beta-COP n = 7; TGN38 n = 5; GM130 n = 11; SEC24C n= 11. Error bars represent SEM.E Primary rat dorsal raphe neurons were cultured After 14 days, cells were infected with lentiviral particles encoding either scramble-shRNA or shRNA directed against MAD2. Five days after infection, cells were fixed in acetone/methanol (1:1) and subjected to immunofluorescence with the indicated primary and secondary antibodies. Confocal images were captured on a Nikon A1 laser scanning confocal microscope at either 20× (Fig. 5 E, left panel) or 60× (Fig. 5 E, right panel) magnification, representative images are shown. Due to imaging conditions the optical section thickness is increased at 20× compared to 60× magnification. Calibration bars in images showing rMAD2 represent arbitrary fluorescence units. Scale bars represent 50 and 10 µm for 20× and 60× magnification, respectively.F Surface and intracellular SERT-signal from scramble- and MAD2-shRNA infected neurons (n = 40 and n = 30, respectively) was quantified within regions of interest (ROIs) using ImageJ software and the resulting ratios compared using an unpaired two-tailed t-test, the p-value was below 0.0001 (n = 4 biological replicates). Error bars represent SD. The right part provides 4 additional representative images of both: scramble-shRNA and MAD2-shRNA expressing serotonergic neurons (SERT: orange, nuclei: blue). Scale bars represent 30 µm.
Figure 2(A) B cell development in Igh∆890/∆890 mice. The relative frequencies of the indicated cell types were determined by flow cytometric analysis of bone marrow cells from Igh∆890/∆890 and Igh+/+ mice at the age of 3-4 weeks, indicating a 1.3-fold increase of pro-B cells and a moderate decrease at subsequent B cell developmental stages in Igh∆890/∆890 mice. Statistical data are shown as mean value with SEM. Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; A, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot (A, corresponds to one mouse.(B) Two-color 3D DNA-FISH analysis of ex vivo sorted pro-B cells of the indicated genotypes with the RP23-340K14 (red) and RP24-275L15 (green) BAC probes (Fig 1A). Representative images are show above. Dot plots (below) show the distances measured between the two DNA signals in individual Igh alleles (2,123 for Rag2-/-, 1,514 for Igh∆890/∆890 Rag2-/- and 502 for Vav-Cre Pax5fl/fl (Pax5∆/∆) Rag2-/-pro-B cells) together with the mean distance (red bar) and SEM determined for each genotype. In total, four independent DNA-FISH experiments were performed. Data information: Statistical data were analyzed by multiple t-tests (unpaired one-way ANOVA (Tukey post-hoc test; B); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.(C, VH gene recombination analysis in ex vivo sorted Igh∆890/∆890 and Igh+/+ pro-B cells, as determined by VDJ-seq experiments (C) The percentages of uniquely identified DJH and VDJH sequences are shown as mean percentage with SEM. Each dot corresponds to an independent VDJ-seq experiments performed with sorted pro-B cells from one mouse. Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot C, corresponds to one mouse.
Figure 3(B) Flow cytometric determination of the ratio of immature IgMb (B6) to IgMa (129) B cells in the bone marrow of Igh∆890(B6)/+(129), IghP4V (B6)/+(129) and control Igh+(B6)/+(129) mice, which were generated by crossing Igh∆890/+, IghP4V/+ and Igh+/+ mice on the C57BL/6 (B6) background with Igh+/+ mice of the 129/Sv (129) strain. The rearranged Igh alleles of the C57BL/6 and 129/Sv strains give rise to the expression of IgMb and IgMa, respectively. The IgMb (B6) to IgMa (129) ratio is shown as mean value with SEM (right). Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; B, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot (B, corresponds to one mouse.(C) Summary of the ratios of immature IgMb (B6) to IgMa (129) B cells, determined for the indicated six mouse strains analyzed and shown as mean values with SEM. The ratio determined for control Igh+(B6)/+(129) B cells was set to 1. Data information: Statistical data were analyzed by multiple t-tests (unpaired one-way ANOVA (Tukey post-hoc test; C, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot C, corresponds to one mouse.(D) Comparison of the VDJ-seq data obtained with pro-B cells of the Igh+/+ (black), Igh∆890/∆890 (white bars) and IghP4V/P4V (gray) genotypes. The different VH genes (horizontal axis) are aligned according to their position in the Igh locus The usage of each VH gene (vertical axis) is shown as percentage of all VDJH and DJH recombination events determined for each pro-B cell type. The relative frequency of each VH gene is shown as mean value with SEM and is based on three independent VDJ-seq experiments for pro-B cells of each genotype.
Figure 4(A) Presence of open chromatin and the active histone marks H3K27ac and H3K4me2 at the V8.8E enhancer in pro-B cells. The location of the VH8-8 gene, V8.8E enhancer (dashed line) and PAIR5 element are shown together with the mm9 genomic coordinates of mouse chromosome 12. The VH8-8 gene with 500 bp of its upstream and downstream sequences (indicated by violet overlay) was inserted with the Floxin method at the deletion point of the Igh∆890 allele to generate the IghV8-8 allele
Figure 5(B, Flow-cytometric analysis (B) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/P4GV (dark green, n=16), IghP4GV/+ (light green, n=12) and control Igh+/+ (black, n=13) mice.C) geometric mean fluorescence intensity measurements (MFI, C) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/P4GV (dark green, n=16), IghP4GV/+ (light green, n=12) and control Igh+/+ (black, n=13) mice. Geometric mean fluorescence intensity measurements (C) are shown as mean values with SEM.G) Absence of GFP expression in DN (CD4-CD8-) and DP (CD4+CD8+) T cells, geometric mean fluorescence intensity measurements (G) of GFP expression in thymocytes from the indicated mice. Geometric mean fluorescence intensity measurements (G) are shown as mean value with SEM based on 12 (Igh+/+), 11 (IghP4GV/+) or 12 (IghP4GV/P4GV) independent replicates.
Figure 6(A, B) Flow-cytometric analysis (A) and geometric mean fluorescence intensity measurements (MFI, B) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/+ (green, n=6), IghP4∆Pax5GV/+ (yellow, n=5), IghP4∆CtcfGV/+ (brown, n=4) and control Igh+/+ (black, n=4) mice. Geometric mean fluorescence intensity measurements (B) are shown as mean value with SEM. Data information: Each dot corresponds to one mouse.(C, D) Activation of the PAIR4-V8.7E module in thymocytes upon loss of CTCF binding at PAIR4, as shown by flow-cytometric analysis (C) and geometric mean fluorescence intensity measurements (MFI, D) of GFP expression in DN, DP, CD4+ and CD8+ thymocytes of IghP4∆CtcfGV/+ (light brown, n=5) and IghP4∆CtcfGV/P4∆CtcfGV (dark brown, n=6) mice compared with control Igh+/+ (black, n=7) mice. Geometric mean fluorescence intensity measurements (D) are shown as mean value with SEM. Data information: Each dot corresponds to one mouse.
Figure 7(B, C) Flow-cytometric analysis (B) and geometric mean fluorescence intensity measurements (MFI, C) of YFP expression are shown for MPPs (LSK), LMPPs, ALPs and BLPs from the bone marrow of Rosa26LSL-YPF/+ IghCμCre/+ (violet) and control Rosa26LSL-YPF/+ Igh+/+ (black) mice. Geometric mean fluorescence intensity measurements (B) are shown as mean value with SEM and have been normalized by setting the mean value obtained with Igh+/+ cells to 1. Data information: Each dot corresponds to one mouse.Flow-cytometric analysis (E) of GFP expression are shown for MPPs (LSK), LMPPs, ALPs, BLPs, pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghEμG/+ (light blue), IghEμG/EμG (dark blue) and control Igh+/+ (black) mice.
Figure 1A) Perturbation in the free energy of PKD2L1 opening (∆G°) and difference in closure rates (∆τclose) inducted by substitutions within the pore module. Error bars = S.D as estimated by the propagation of error equation, as descried in statistics subsection.
Figure 2B) Exemplar tail currents from mutant channels where the energetics of opening are disassociated with the rate of channel closure- mutations which cause slower closure (+∆τclose) while also increasing the free energy opening (+∆G°).
Figure 3D) Comparing voltage-dependent opening based on normalized tail currents measured from WT, single and double mutant channels. Top, single and double mutant channels including mutations to the outer and inner gate. Middle and bottom, control groups comparing tail currents from non-gate S4-S5 linker mutations (D476A and G479A) and those from the outer and inner gates. Error bars indicate standard deviation for as estimated by the propagation of error equation, as descried in statistics subsection. Number cells tested (N) are indicated in parentheses for each group.E) The coupling energy (∆∆G°) resulting from the mutant cycle analysis resulting from the data shown in D. Error bars indicate standard deviation for as estimated by the propagation of error equation, as descried in statistics subsection. Number cells tested (N) are indicated in parentheses for each group.
Figure 4B) Summary of the change in voltage-dependent inactivation (∆V1/2 Inact.) of pore mutant channels. ∆V1/2 Inact., are plotted relative to the pore module topology of the channel.C) Voltage-dependent inactivation of test pulse tail currents recorded from WT, single and double mutant channels.
Figure 5C) Exemplar whole cell currents (Top) at the zero, three and seven minute time points. Current records (Bottom) from cells expressing the F514C:P538C and treated with extracellular 100 μM 1,2-methanediyl bismethanethiosulfonate (M2M).D) Box (S.E.M.) and whisker (S.D.) plots comparing tail currents normalized to their initial amplitude at 3 minute (Left) and 7-minute time points (Right). N = 6 cells per group. Results from each individual cells are indicated by grey open circles and the average for the channel is indicated by a horizontal line. P-values results from a one-way ANOVA statistical analysis is shown above the plots. Dashed lines the level normalcy, no change in Itail relative to time point zero.